Supporting Information� Copyright Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, 2014

Tuning the Guest-Binding Ability of a Helically Folded Capsule byIn Situ Modification of the Aromatic Oligoamide Backbone

Guillaume Lautrette,[a] Christophe Aube,[b] Yann Ferrand,[a] Muriel Pipelier,[b] Virginie Blot,[b]

Christine Thobie,[b] Brice Kauffmann,[c] Didier Dubreuil,*[b] and Ivan Huc*[a]

chem_201303929_sm_miscellaneous_information.pdf

S1

Supporting Information

Tuning the guest binding ability of a helically folded capsule by in situ modification of its aromatic oligoamide backbone

Guillaume Lautrette, [a] Christophe Aube,[b] Yann Ferrand,[a] Muriel Pipelier, Virginie Blot,[b]

Christine Thobie,[b] Brice Kauffmann,[c] Didier Dubreuil*,[b] and Ivan Huc*,[a]

Table of contents

Page S2 Synthetic schemes

Page S3 Materials and methods

Page S4 NMR titrations

Page S9 Solution structure determination

Page S23 Crystallography

Page S25 Experimental section

Page S30 References

Page S31 NMR spectra

S2

Synthetic schemes

Scheme S1. Synthesis of capsules 2 and 3 : (a) Acetic anhydride, (iPr)2NEt, CHCl3, 60°C ; (b) TFA, CHCl3,

rt ; (c) PyBop, (iPr)2NEt, CHCl3, 45°C.

S3

Materials and methods

Nuclear Magnetic Resonance

NMR spectra were recorded on 3 different NMR spectrometers: (1) an Avance II NMR spectrometer

(Bruker Biospin) with a vertical 7.05T narrow-bore/ultrashield magnet operating at 300 MHz for 1H

observation, 282 MHz for 19F observation and 75 MHz for 13C observation by means of a 5-mm direct BBO

H/X probe with Z gradient capabilities; (2) a DPX-400 NMR spectrometer (Bruker Biospin) with a vertical

9.4T narrow-bore/ultrashield magnet operating at 400 MHz for 1H observation by means of a 5-mm direct

QNP 1H/13C/31P/19F probe with gradient capabilities; (3) an Avance III NMR spectrometer (Bruker Biospin)

with a vertical 16.45T narrow-bore/ultrashield magnet operating at 700 MHz for 1H observation by means of

a 5-mm TXI 1H/13C/15N probe with Z gradient capabilities. Chemical shifts are reported in parts per million

(ppm, ) relative to the 1H residual signal of the deuterated solvent used. 1H NMR splitting patterns with

observed first-order coupling are designated as singlet (s), doublet (d), triplet (t), or quartet (q). Coupling

constants (J) are reported in hertz. Samples were not degassed. Data processing was performed with Topspin

2.0 software.

Crystallography

Data collections were performed at the IECB X-ray facility (UMS 3033 CNRS) on a RIGAKU

MM07 rotating anodes at the copper kα wavelength at 213K (receptor 3). The crystal was mounted on cryo-

loops after quick soaking on Paratone—N oil from Hampton research and flash-frozen. Crystal structure was

solved by the ab-initio method with dual-space recycling using the largest E-values implemented in

SHELXD and refined using SHELXL. Full-matrix least-squares refinement was performed on F2 for all

unique reflections, minimizing w(Fo2- Fc2)2, with anisotropic displacement parameters for non-hydrogen

atoms. Hydrogen atoms were included in idealized positions and refined with a riding model, with Uiso

constrained to 1.2 Ueq value of the parent atom (1.5 Ueq when CH3). PLATON/SQUEEZE was used to treat

two badly disordered dichloromethane molecules in the asymmetric unit. These were removed from the

model. SQUEEZE reports that the volume of total potential solvent accessible void is 1087.2 Å3 in the unit

cell and the number of electrons within the void is reported to be 263 electrons/cell.

S4

NMR Titrations

Determination of Ka through NMR integration:

For the equilibrium shown in Eq. 1, the association constant Ka of the receptor is given by Eq. 2.

(1)

(2)

where: [HG] = complex concentration; [H] = host concentration; [G] = guest concentration

Alternatively, (3)

where: VT = total volume of the sample; nH = number of moles of host (etc.)

From mass balance, HGHH nnn 0 (4)

HGGG nnn 0 (5)

where: nH0 = initial number of moles of host; nG0 = number of moles of guest added to the sample

Substituting equations (4) and (5) into (3), (6)

From integration of the NMR spectrum it is possible to obtain the fraction of bound host molecules, x (Eq.

7).

0H

HG

n

nx (7)

Using Eq. 7 to eliminate nHG from Eq. 6, (8)

H + G HG

GH

HGKa

GH

THGa

nn

VnK

HGGHGH

THGa

n-nn-n

VnK

00

)nx()n(x-)n(x-n

VxK

0000 H2

GHG

Ta

S5

Figure S1. Part of the 400MHz 1H NMR spectra of 2 (2mM) in CDCl3 at: A. 303 K; B. 298 K; C. 293 K; D.

288 K; E. 283 K; F. 278 K; G. 273 K; H. 268 K; I. 263 K. Signals of the anti-anti capsule are marked with

empty squares.

Figure S2. Part of the 400MHz 1H NMR spectra of 3 (2mM) in CD2Cl2 at: A. 308 K; B. 303 K; C. 298 K;

D. 293 K; E. 288 K; F. 283 K; G. 278 K; H. 273 K; I. 268 K; J. 263 K. Signals of the anti-anti and of the

syn-syn capsule are marked with empty and black squares, respectively. The black triangle denotes the

resonance of syn-syn NH of pyrrole.

S6

Figure S3. Representative 400 MHz NMR spectra of 1 (1 mM) in a mixture of CDCl3:d6-DMSO (99:1

vol/vol) at 298 K titrated with D/L-malic acid: A. 0 equiv., B. 0.5 equiv., C. 1 equiv., D. 2 equiv., E. 5 equiv..

Amide signals of the empty host are marked with empty white circles. Amide signals of the match host-guest

complex and of the mismatch host-guest complex (i.e. diastereoisomer) are marked with black circles and

black triangles, respectively. At low field (~ 14 ppm), one can see the two different carboxylic acid protons

of malic acid (diamond). Ka = 9250 L.mol-1

Figure S4. Representative 300 MHz NMR spectra of 3 (1 mM) in a mixture of CDCl3:d6-DMSO (99:1

vol/vol) at 298 K titrated with D/L-tartaric acid: A. 0 equiv., B. 0.25 equiv., C. 1 equiv., D. 1.5 equiv., E. 3

equiv.. Amide signals of the empty host and of the host–guest complex are marked with empty white and

black circles, respectively. At low field (~ 13 ppm), one can see carboxylic acid protons of the bound tartaric

acid (diamond). Ka = 16000 L.mol-1

S7

Figure S5. Representative 300 MHz NMR spectra of 3 (1 mM) in a mixture of CDCl3:d6-DMSO (90:10

vol/vol) at 298 K titrated with D/L-tartaric acid: A. 0 equiv., B. 1 equiv., C. 4 equiv., D. 8 equiv., E. 16

equiv.. Amide signals of the empty host and of the host–guest complex are marked with empty white and

black circles, respectively. At low field (~ 13ppm), one can see carboxylic acid protons of the bound tartaric

acid (diamond). Ka = 110 L.mol-1

Figure S6. Representative 400 MHz NMR spectra of 3 (1 mM) in a mixture of CDCl3:d6-DMSO (99:1

vol/vol) at 298 K titrated with D/L-malic acid: A. 0 equiv., B. 2 equiv., C. 8 equiv., D. 16 equiv., E. 32 equiv..

Amide signals of the empty host and of the host–guest complex are marked with empty white and black

circles, respectively. Ka = 100 L.mol-1

S8

Figure S7. CD spectra of 3 (200 µM in CHCl3:d6-DMSO 99:1 vol/vol) at 298 K titrated with 10 equiv. of D-

tartaric acid.

S9

Figure S8. 1H NMR spectrum (300 MHz) of 1 left) and 3 (right) in CDCl3 at 298 K.

S10

Figure S9. 1H NMR spectrum (300 MHz) of 1 D/L-tartaric acid (left) in CDCl3/d6-DMSO (99/1 vol/vol) at

298 K. 1H NMR spectrum (300 MHz) of 3 D/L-tartaric acid (right) in CDCl3/d6-DMSO (99/1 vol/vol) at

298 K.

S11

Determination of the structures of 3 and 3 D/L-tartaric in solution by 1D and 2D NMR:

Figure S10. Part of the 400 MHz COSY plot of 3 in CDCl3 at 298 K. The spin systems of pyridine rings

from pyr-pyl-pyr (pyr1, grey), pyrrole rings (Pyl, orange), diaminopyridine rings (pyr2, red), naphthyridine

rings (N, green) and quinoline rings (Q, blue) are connected by color coded full lines.

S12

Figure S11. Parts of the 400 MHz HSQC plot of 3 in CDCl3 at 298 K, showing cross-peaks between directly

bonded hydrogen and carbons. The horizontal scale is that of proton resonances and the vertical scale is that

of carbon resonances.

S13

Figure S12. Parts of the 400 MHz HMBC plot of 3 in CDCl3 at 298 K. The horizontal scale is that of proton

resonances and the vertical scale is that of carbon resonances.

S14

Figure S13. 2D ROESY spectrum (400 MHz) of 3 in CDCl3 at 298 K showing through-space correlations

(intramolecular NOE contacts).

S15

Figure S14. Zoom of 2D NOESY spectrum (400 MHz) of 3 in CDCl3 at 298 K showing intramolecular

correlations (NOE contacts) between the amine group of the pyrrole and the ortho position of the

neighbouring pyridine (pyr1).

S16

Table S1. 1H and 13C chemical shifts for 3 in CDCl3.

1H 13C 1H 13C

pyl-H 6.96 113.8 Q2-H3 7.41 100.9

pyr1-H3 8.09 132.3 Q2-H5 6.59 115.5

pyr1-H4 7.62 136.7 Q2-H6 6.10 126.5

pyr1-H5 6.16 118.2 Q2-H7 8.14 116.2

N1-H3 7.91 98.5 Q3-H3 6.74 98.7

N1-H5 8.66 133.4 Q3-H5 7.61 114.9

N1-H6 8.89 115.5 Q3-H6 6.92 126.4

N2-H3 7.66 98.4 Q3-H7 7.49 115.9

N2-H5 8.09 118.6 NH-1 10.56

N2-H6 8.21 112.6 NH-2 10.69

pyr2-H3 7.12 107.7 NH-3 9.78

pyr2-H4 6.63 138.0 NH-4 9.38

pyr2-H5 6.99 107.5 NH-5 11.53

Q1-H3 6.04 97.0 NH-6 11.73

Q1-H5 7.59 115.7 NH-Ac 8.53

Q1-H6 6.90 127.2 CH3Ac 1.32 23.9

Q1-H7 7.99 116.5 NH-Pyl 5.91

S17

Figure S15. Part of the 700 MHz COSY plot of 3 D/L-tartaric acid in CDCl3:d6-DMSO (99:1 vol/vol) at

298 K. The spin systems of pyridine rings from pyr-pyl-pyr (pyr1, grey), pyrole rings (Pyl, orange),

diaminopyridine rings (pyr2, red), naphthyridine rings (N, green) and quinoline rings (Q, blue) are connected

by color coded full lines.

S18

Figure S16. Parts of the 700 MHz HSQC plot of 3 D/L-tartaric acid in CDCl3:d6-DMSO (99:1 vol/vol) at

298 K, showing cross-peaks between directly bonded hydrogen and carbons. The horizontal scale is that of

proton resonances and the vertical scale is that of carbon resonances.

S19

Figure S17. Parts of the 700 MHz HMBC plot of 3 D/L-tartaric acid in CDCl3:d6-DMSO (99:1 vol/vol) at

298 K. The horizontal scale is that of proton resonances and the vertical scale is that of carbon resonances.

S20

Figure S18. 2D ROESY spectrum (700 MHz) of 3 D/L-tartaric acid in CDCl3:d6-DMSO (99:1 vol/vol) at

298 K showing through-space correlations (intra and intermolecular NOE contacts).

S21

Figure S19. Zoom of 2D ROESY (700MHz) spectrum (top) and of COSY (700MHz) spectrum (bottom) of

3 D/L-tartaric acid in CDCl3:d6-DMSO (99:1 vol/vol) at 298 K showing intramolecular correlations (NOE

contacts) and assignment of tartaric acid in the complex, respectively.

S22

Table S2. 1H and 13C chemical shifts for 3 D/L tartaric acid in CDCl3:d6-DMSO (99:1 vol/vol).

1H 13C 1H 13C

Pyl-H 7.22 113.1 Q2-H5 6.60 118.6

Pyr1-H3 7.89 115.8 Q2-H6 7.69 136.5

Pyr1-H4 6.91 126.6 Q2-H7 8.08 118.2

Pyr1-H5 7.59 97.9 Q3-H3 7.25 99.7

N1-H3 7.92 99.3 Q3-H5 6.68 115.4

N1-H5 9.06 115.6 Q3-H6 6.22 126.3

N1-H6 8.71 134.5 Q3-H7 8.09 115.7

N2-H3 7.58 115.4 NH-1 11.56

N2-H5 8.22 112.9 NH-2 10.63

N2-H6 8.00 131.6 NH-3 10.15

Pyr2-H3 6.99 106.9 NH-4 9.59

Pyr2-H4 6.56 137.4 NH-5 11.68

Pyr2-H5 7.26 107.6 NH-6 11.80

Q1-H3 5.92 96.2 NH-Ac 8.53

Q1-H5 7.48 115.5 NH-Pyl 6.69

Q1-H6 6.91 126.4 COOH 13.20

Q1-H7 7.54 112.8 CHOH 4.41 73.7

Q2-H3 6.84 98.7 CHOH 4.80

S23

Crystallography

Figure S20. Side view of the crystal structure of 3: a) in CPK representation; b) in tube representation. Tube

representations of the solid-state structures of 3: c) top view and d) slice of the capsule showing the N2-pyr-

pyl-pyr-N2 segment from above of the P-helix of 3 (Q3PN2-pyr-pyl-pyr-N2PQ3 with an anti-anti

conformation of the pyrrole). Isobutyl side chains and included solvent molecules have been removed for

clarity.

S24

Table S3: Crystal data and structure refinement for capsule 3

Name Receptor 3

Formula C176 H181 Cl19.50 N33 O25

M 3849.80

Crystal system triclinic

Space group P-1

a/Å 18.261(6)

b/Å 24.751(9)

c/Å 25.506(7)

α/° 114.19(3)

β/° 97.69(3)

γ/° 102.08(3)

U/Å3 9967(6)

T /K 213(2)

Z 2

ρ/g cm–1 1.283

Shape and color Colorless needles

size (mm) 0.1x0.1x0.05

λ/ Å 1.54178

μ/mm-1 3.028

Total reflections 74035

Unique data 16832

Rint 0.0808

parameters/restraints 2290/3

R1, wR2 0.1654/ 0.4267

goodness of fit 1.586

CCDC # 964832

S25

Experimental section.

General. All reactions were carried out under a dry nitrogen atmosphere. Commercial reagents were

purchased from Sigma-Aldrich or Alfa-Aesar and were used without further purification unless otherwise

specified. Chloroform, diisopropylethylamine (DIPEA) were distilled from calcium hydride (CaH2) prior to

use. Reactions were monitored by thin layer chromatography (TLC) on Merck silica gel 60-F254 plates and

observed under UV light. Column chromatographies were carried out on Merck GEDURAN Si60 (40-63

μm). Proton nuclear magnetic resonance (1H NMR) spectra were recorded in deuterated solvents on 300 and

400 MHz spectrometers. Chemical shifts are reported in parts per million (ppm, ) relative to the signal of

the NMR solvent used. 1H NMR splitting patterns with observed first-order coupling are designated as

singlet (s), doublet (d), triplet (t), or quartet (q). Coupling constants (J) are reported in hertz. Splitting

patterns that could not be interpreted or easily visualized are designated as multiplet (m) or broad (br). 13C

NMR spectra were recorded on 300 or 400 MHz spectrometers. Chemical shifts are reported in ppm ()

relative to carbon resonances of the NMR solvent. Mass spectra (MS) were obtained using matrix-assisted

laser desorption/ionization (MALDI) or were measured on a DSQ Thermoelectron apparatus by electronic

impact (70eV). High Resolution Mass Spectra (HRMS) were measured by electronic impact or by chemical

ionisation on quadripolar spectrometers KRATOS MS 80RF or Micromasse Q T of 1. They were equally

measured by MALDI-TOF in positive ionization mode on spectrometer Autoflex III of Bruker in the service

“Biopolymères, Interactions, Biologie Structurale” of INRA (Institut National de la Recherche

Agronomique). The used matrices were DHB (2,5-DiHydroxyBenzoic acid) or DCTB (T-2-(3-(4-t-

Butylphenyl)-2-methyl-2-propenylidene)malononitrile). Melting points were measured on a RCH

microscope (C. Reichert) with a heating plate KOFLER or by a Tottoli microscope (Büchi) with the aid of

capillaries. FT-IR spectra were measured on a Bruker Vector 22 FT-IR spectrometer between 500 and 4000

cm-1 by KBr pellets. Cyclic voltammetry experiments and preparative electrolysis were performed using a

potentiostat-galvanostat SP 300 (Biologic) controlled by the EC-Lab software. In cyclic voltammetry, a

conventional three-electrode system was used with a glassy carbon working electrode, a saturated calomel

reference electrode (SCE) and a platinum wire counter electrode. Preparative electrolysis were carried out

under cathodic potential (Et) in a concentric cylindrical cell with two compartments separated by a glass frit.

A mercury pool electrode (diameter 4.7 cm) or reticulated vitreous carbon (HF 1077-BAS) was used as the

cathode, calomel saturated electrode as reference and platinum plate as the anode. In the cathodic

compartment, the solution is deoxygenated before electrochemical measures.

S26

Capsule 2. Diacid 5 (0.036 mmol, 0.012 g) and hexamer amine 10 (0.072 mmol, 0.099 g) were

dissolved in dry chloroform (2 mL). Then, DIPEA (0.18 mmol, 0.032 mL) and PyBop (0.18 mmol, 0.094 g)

were added at RT and the reaction mixture was heated at 45°C for 12 hours. Then, the solvents were

removed under reduced pressure and the residue was purified by flash chromatography (SiO2) eluting with

EtOAc:cyclohexane (20:80 vol/vol) and by precipitation from minimum amount of MeOH to obtain 2 as a

light yellow solid (70 %, 0.075 g). 1H NMR (300 MHz, CDCl3) ppm = 11.56 (s, 2H); 11.36 (s, 2H); 10.64

(s, 2H); 9.92 (s, 2H); 9.87 (s, 2H); 8.89 (s, 2H); 8.82 (d, 3J = 9.0, 2H); 8.68 (d, 3J = 8.9, 2H); 8.59 (d, 3J =

8.8, 2H); 8.45 (s,2H); 8.37 (s, 2H); 8.27 (m, 8H); 8.18 (d, 3J = 9.0, 2H); 7.99 (d, 3J = 8.7, 2H); 7.87 (t, 3J =

7.8, 2H); 7.57 (m, 6H); 7.40 (m, 8H); 7.19 (m, 4H); 6.93 (t, 3J = 8.0, 2H); 6.72 (m, 8H); 6.3 (m, 4H); 6.01 (t,

3J = 7.9, 2H); 4.13 (m, 8H); 3.99 (t, 3J = 7.3, 2H); 3.86 (m, 5H); 3.72 (m, 5H); 2.82 (m, 3H); 2.36 (m, 7H);

1.28 (m, 36H); 1.10 (m, 12H); 0.57 (d, 3J = 6.6, 6H); 0.44 (d, 3J = 6.8, 6H). 13C NMR (100 MHz, CDCl3)

ppm = 166.8; 164.0; 163.4; 163.2; 163.1; 163.0; 162.3; 161.5; 161.4; 160.5; 159.2; 154.9; 154.7; 153.7;

153.6; 153.1; 152.0; 151.5; 151.1; 150.7; 150.1; 148.9; 148.2; 148.1; 146.8; 139.7; 138.4; 137.6; 137.1;

136.5; 134.2; 134.1; 133.6; 133.1; 126.8; 126.5; 125.9; 124.6; 123.9; 123.7; 122.4; 121.6; 121.4; 116.7;

116.6; 116.2; 116.0; 115.6; 114.6; 114.4; 114.1; 113.7; 108.8; 107.7; 100.7; 98.4; 98.1; 96.9; 76.0; 75.8;

75.2; 75.2; 28.5; 28.4; 28.2; 28.1; 27.6; 23.8; 19.6; 19.5; 19.5; 19.4; 19.1; 18.5. MS (MALDI): m/z calcd for

C166H160N34O24 [M+H]+: 3015.2457 found 3015.10.

Capsule 3. Diacid 4 (0.083 mmol, 0.026 g) and hexamer amine 10 (0.150 mmol, 0.202 g) were

dissolved in dry chloroform (4 mL). Then, DIPEA (0.30 mmol, 0.05 mL) and PyBop (0.23 mmol, 0.120 g)

were added at RT and the reaction mixture was heated at 45°C for 24 hours. Then, the solvents were

removed under reduced pressure and the residue was purified by flash chromatography (SiO2) eluting with

EtOAc:DCM (10:90 vol/vol) and by precipitation from minimum amount of MeOH to obtain 3 as a yellow

solid (44 %, 0.098 g). 1H NMR (400 MHz, CDCl3) ppm = 11.73 (s, 2H); 11.53 (s, 2H); 10.69 (s, 2H);

10.56 (s, 2H); 9.78 (s, 2H); 9.38 (s,2H); 8.89 (d, 3J = 8.8, 2H); 8.66 (d, 3J = 8.8, 2H); 8.53 (s,2H); 8.21 (d, 3J

= 8.8, 2H); 8.11 (m, 6H); 7.99 (d, 3J = 7.4, 2H); 7.91 (s, 2H); 7.62 (m, 8H); 7.49 (d, 3J = 7.4, 2H); 7.41 (s,

2H); 7.12 (d, 3J = 7.7, 2H); 6.94 (m, 10H); 6.63 (t, 3J = 7.8, 2H); 6.59 (d, 3J = 8.2, 2H); 6.16 (d, 3J = 7.7, 2H);

6.10 (t, 3J = 7.8, 2H); 6.04 (s, 2H); 5.91 (br, 1H); 4.44 (t, 3J = 8.0, 2H); 4.27 (m, 6H); 3.89 (t, 3J = 6.1, 2H);

S27

3.74 (t, 3J = 8.0, 2H); 3.60 (t, 3J = 6.6, 2H); 3.53 (t, 3J = 7.9, 2H); 2.99 (m, 4H); 2.46 (m, 5H); 2.28 (m, 5H);

1.31 (m, 36H); 1.11 (m, 18H); 0.60 (d, 3J = 6.4, 6H); 0.13 (d, 3J = 6.5, 6H). 13C NMR (100 MHz, CDCl3)

ppm = 166.9; 164.3; 164.1; 163.4; 163.2; 163.0; 163.0; 162.9; 161.9; 161.7; 161.3; 159.9; 157.7; 154.7;

153.9; 153.1; 152.5; 150.6; 150.4; 149.2; 148.9; 148.2; 147.4; 147.4; 138.6; 138.0; 137.8; 136.7; 136.5;

134.6; 134.0; 133.4; 133.2; 132.3; 131.6; 127.2; 126.5; 126.4; 122.8; 121.7; 121.2; 118.6; 118.2; 116.5;

116.2; 115.9; 115.7; 115.5; 115.3; 114.9; 113.8; 113.7; 112.6; 107.7; 107.5; 100.9; 98.7; 98.5; 98.4; 97.0;

76.1; 76.8; 75.3; 75.2; 75.1; 28.6; 28.5; 28.4; 28.2; 27.4; 23.9; 19.6; 19.5; 19.4; 19.4; 19.0; 18.1. MS

(MALDI): m/z calcd for C166H161N34O24 [M+H]+: 3002.2504 found 3002.05.

In situ chemical reduction of pyridazine casule 2 to give pyrrole capsule 3 :

To a solution of capsule pyridazine 2 (4.98 mmol, 0.015 g,) in glacial acetic acid (1 mL) heated at reflux was

added activated zinc (99 mmol, 0.0065 g) the reaction was stirred at this temperature for 4 hours. After

cooling to R.T., the mixture was filtered through a pad of celite which was washed with MeOH (10 mL) and

concentrated. After flash chromatography (SiO2) eluting with PE:EtOAc:DCM 60:30:10 (vol/vol), the

capsule 3 was obtained as a yellow powder (50 %, 0.007 g).

Compound 4. Dioctyl 6,6’-(1H-pyrrol-2,5-diyl)bis-2-pyridinecarboxylate 7 (0.11 mmol, 0.06 g) was

dissolved in MeOH (5 mL) and H2O (1 mL) and was added a solution of KOH (0.45 mmol, 0.025 g)

dissolved in H2O (0.5 mL per mmol of starting material). After 4 hours at reflux, the crude was concentrated

and the residue was treated with H2O and aqueous 2N HCl solution until a pH value of 3 was obtained. After

addition of MeOH, a precipitate was formed. The solid was washed consecutively with MeOH, H2O and

Et2O and dried under vacuum. The compound 4 was obtained as a yellow powder (85%, 0.029 g). m.p.

301°C; 1H NMR (300 MHZ, DMSO-d6) = 13.08 (br, 2H, OH); 11.90 (br, 1H, NH); 8.11 (d, 3J = 7.8, 2H);

8.01 (t, 3J = 7.8, 2H); 7.87 (d, 3J = 7.8, 2H); 7.07 (s, 2H); 13C NMR (75 MHz, DMSO-d6): = 165.8; 149.7;

147.1; 138.5; 132.2; 122.3; 121.5; 111.3; IR: = 3415 cm-1 (NH), 1732 cm-1 (C=O); MS (EI, 70eV): m/z

(%): 309 (40) [M+], 265 (41) [M+ - (2OH)], 221 (100) [M+ - (2CO2H)], HRMS (ESI+): calcd. for

C16H12N3O4 [M + H]+ 309.0750; found 309.0751.

Compound 6. 6,6’-(pyridazin-3,6-diyl)bis-2-pyridylcarboxylic acid 51 (2.79 mmol, 0.90 g) was

dissolved in SOCl2 (8 mL) and the mixture was refluxed during 18 hours. After elimination of thionyl

chloride by distillation under vacuum, the obtained dipicolinyl chloride was dissolved in DCM (5 mL). After

S28

cooling to 0 °C, triethylamine (16.77 mmol, 2.3 mL) and the desired alcohol (8.38 mmol, 1.3 mL) were

successively added and the mixture was stirred at R.T. under inert atmosphere until total consumption of the

corresponding dipicolinyl chloride. The mixture was diluted in DCM, washed with water, dried over Na2SO4

and concentrated. After flash chromatography (SiO2) eluting with DCM:MeOH (95:5 vol/vol), the compound

6 was obtained as a white powder (88%, 1.3 g). m.p. 89°C; 1H NMR (300 MHZ, CDCl3) ppm = 8.94 (d, 3J

= 7.8, 2H); 8.85 (s, 2H); 8.20 (d, 3J = 7.8, 2H); 8.05 (t, 3J = 7.8, 2H); 4.44 (t, 3J = 6.8, 4H, CH2); 1.89 - 1.77

(m, 4H, CH2); 1.51 - 1.29 (m, 20H, CH2); 0.87 (t, 3J = 6.6, 6H, CH3); 13C NMR (75 MHz, CDCl3) ppm

=165.1; 157.9; 153.7; 148.4; 138.3; 126.1; 125.8; 124.7; 66.3; 31.9; 29.5; 29.4; 28.8; 26.1; 22.8; 14.2; IR:

= 1716 cm-1 (C=O); MS (EI, 70eV): m/z (%): 546 (100) [M+], 433(20) [M+ - (C8H17)], 232 (58) [M+ -

(2C9H17O2)], HRMS (ESI+): calcd. for C32H42N4NaO4 [M + Na]+ 569.3098; found 569.3096.

Compound 7. A solution of dioctyl 6,6’-(pyridazin-3,6-diyl)bis-2-pyridinecarboxylate 6 (0.44

mmol, 0.242 g) was dissolved in THF/acetate buffer/CH3CN (5/4/1, 100 mL), introduced into the cathodic

compartment of the electrochemical cell and reduced at Ew = -1.15 V/SCE. Prior to and during electrolysis at

Ew, the catholyte was deaerated with argon and stirred magnetically. The course of the reaction was followed

by cyclic voltammetry in the cathodic compartment and the electrolysis was stopped when the consumption

of the starting substrate was completed. Then the solvents were partially evaporated, the aqueous phase was

neutralized by addition of aqueous saturated Na2CO3 solution and extracted with DCM. The combined

organic layers were dried (Na2SO4) and concentrated. After flash chromatography (SiO2) eluting with

PE:EtOAc (75:25 vol/vol), the compound 7 was obtained as a yellow powder (51%, 0.120 g). m.p. 68°C; 1H

NMR (300 MHZ, CDCl3) ppm = 12.12 (br, 1H, NH); 7.85 (dd, J = 7.8 and 1.9, 2H); 7.79 (t, J = 7.8, 2H);

7.73 (dd, J = 7.8 and 1.9, 2H); 6.81 (d, J = 2.5, 2H); 4.42 (t, J = 6.8, 4H, CH2); 1.90 - 1.80 (m, 4H, CH2),

1.49 - 1.25 (m, 20H, CH2); 0.87 (t, J = 6.6, 6H, CH3); 13C NMR (75 MHz, CDCl3) ppm = 165.6; 150.9;

147.7; 137.3; 133.7; 122.1; 121.9; 110.1; 66.3; 31.9; 29.5; 29.4; 28.8; 26.1; 22.8; 14.2; IR: = 3431 cm-1

(NH), 1728 cm-1 (C=O); MS (EI, 70eV): m/z (%): 533 (100) [M+], 420 (7) [M+ - (C8H17)], 219 (33) [M+ -

(2C9H17O2)], HRMS (ESI+): calcd. for C32H44N3O4 [M + H]+ 534.3326; found 534.3321.

AcNH-Q3PN2-NHBoc (9). To a solution of H2N-Q3PN2-NHBoc 82 (0.23 mmol, 0.33 g) in dioxane

(12 mL) was added acetic anhydride (2.3 mmol, 0.2 mL) and (2.3 mmol, 0.4 mL). After 12 hours at 60°C,

the reaction mixture was let at RT. The volatiles were removed under reduced pressure to give a solid which

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was dissolved in dichloromethane and washed with a saturated solution of NaHCO3, distilled water and then

with brine. The organic layers were dry over Na2SO4 then the volatiles were removed under reduce pressure

to give 9 as a pale yellow solid (96%, 0.33 g). 1H NMR (300 MHz, CDCl3) ppm = 12.41 (s, 1H); 12.25 (s,

1H); 11.03 (s, 1H); 10.36 (s, 1H); 9.91 (s, 1H); 9.84 (s,1H); 9.45 (d, 3J = 7, 1H); 9.11 (s, 1H); 8.96 (s, 1H);

8.76 (m, 2H); 8.62 (m, 3H); 8.18 (d, 3J = 7.8, 1H); 8.09 (d, 3J = 7.6, 1H); 7.87 (m, 3H); 7.74 (m, 4H); 7.31 (s,

H); 7.21 (t, 3J = 8.2, 1H); 6.98 (d, 3J = 7.4, 1H); 6.87 ( s,1H); 6.81 (t, 3J = 7.9, 1H); 4.14 (m, 7H); 3.87 (m,

3H); 2.39 (m, 5H); 1.91 (m, 3H); 1.83 (s, 9H); 1.19 (m, 24H); 0.71 (d, 3J = 6.5, 3H); 0.52 (d, 3J = 6.7, 3H).

13C NMR (75 MHz, CDCl3) ppm = 167.1; 164.7; 164.2; 164.0; 163.9; 163.6; 163.3; 161.7; 161.5; 160.2;

156.9; 155.0; 154.3; 153.9; 153.6; 153.1; 152.1; 151.3; 150.5; 149.3; 148.4; 147.9; 140.2; 138.6; 138.3;

136.9; 134.3; 134.3; 134.1; 133.5; 133.4; 127.7; 126.8; 123.0; 122.2; 122.0; 117.7; 117.4; 116.7; 116.2;

115.9; 115.9; 114.8; 114.8; 114.2; 109.4; 108.6; 102.7; 99.1; 98.4; 98.2; 98.0; 81.7; 77.3; 75.8; 75.7; 75.5;

75.3; 28.7; 28.3; 28.2; 28.0; 24.5; 19.5; 19.4; 19.3; 19.3; 19.2; 18.6. HRMS (MALDI): m/z calcd for

C80H85N15O13 [M+Na]+ 1486.63490 found 1486.63151.

AcNH-Q3PN2-NH2 (10). Trifluoroacetic acid (0.2 mL) was added drop wise to a solution of 9 (0.07

mmol, 0.1 g) in 1 mL of chloroform under nitrogen at RT. Then, the resultant mixture was stirred at RT for 4

hours. The volatiles were removed under reduced pressure to give a solid which was dissolved in

dichloromethane and washed with a saturated solution of NaHCO3, distilled water and then with brine. The

organic layers were dry over Na2SO4 then the volatiles were removed under reduce pressure to give the

amine derivative 10 as pale yellow solid (97%, 0.093 g). 1H NMR (300 MHz, CDCl3) ppm = 12.35 (s, 1H);

12.22 (s, 1H); 11.36 (s, 1H); 9.79 (s, 1H); 9.78 (s, 1H); 9.14 (d, 3J = 7.3, 1H); 9.08 (s, 1H); 8.77 (m, 2H);

8.64 (s, 1H); 8.60 (d, 3J = 7.4, 1H); 8.30 (d, 3J = 8.9, 1H); 8.24 (d, 3J = 7.6, 1H); 7.84 (m, 5H); 7.71 (t, 3J =

8.0, 1H); 7.54 (s, 1H); 7.28 (m, 2H); 7.21 (t, 3J = 8.1, 1H); 7.03 (d, 3J = 8.2, 1H); 6.89 (m,2H); 6.75 (t, 3J =

8.1, 1H); 6.31 (br, 2H); 4.59 (br, 1H); 4.20 (m, 3H); 4.05 (m, 3H); 3.91 (d, 3J = 6.4, 2H); 3.77 (t, 3J = 8.5,

1H); 2.36 (m, 5H); 1.87 (s, 3H); 1.19 (m, 24H); 0.70 (d, 3J = 6.1, 3H); 0.48 (d, 3J = 6.3, 3H). 13C NMR (75

MHz, CDCl3) ppm = 167.4; 164.6; 164.1; 164.0; 163.8; 163.6; 163.3; 162.5; 161.8; 160.6; 156.5; 154.8;

154.2; 153.5; 151.2; 150.7; 150.6; 149.7; 148.9; 147.9; 140.4; 138.7; 138.2; 137.1; 134.5; 134.4; 134.2;

133.7; 132.6; 128.0; 127.0; 126.8; 123.0; 122.2; 122.1; 117.7; 117.2; 116.7; 116.4; 116.0; 116.0; 115.0;

114.6; 113.8; 111.0; 109.9; 109.0; 102.1; 99.1; 98.7; 98.1; 77.4; 76.0; 75.6; 75.4; 28.5; 28.4; 28.2; 27.9; 24.5;

19.6; 19.5; 19.4; 19.3; 18.6. HRMS (ES+): m/z calcd for C75H77N15O11 [M+H]+: 1364.60052 found

1365.59850.

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References

1. Y. Ferrand, A. M. Kendhale, B. Kauffmann, A. Grélard, C. Marie, V. Blot, M. Pipelier, D. Dubreuil, I.

Huc, J. Am. Chem. Soc. 2010, 23, 7858.

2. Y. Ferrand, C. Nagula, A. M. Kendhale, C. Aube, B. Kauffmann, A. Grelard, M. Laguerre, D. Dubreuil,

I. Huc, J. Am. Chem. Soc. 2012, 134, 11282-11288.

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1H NMR spectra of all relevant synthetic intermediates and title compounds.

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